The C-Terminal Penta-Peptide Repeats of Major Royal Jelly Protein 3 Ameliorate the Progression of Inflammation in Vivo and in Vitro

Abstract

Royal jelly (RJ) has been used as a functional foodstuff and in cosmetics for many years. RJ contains various molecules, including major royal jelly proteins (MRJPs), and affords a number of health benefits such as anti-inflammatory activity. As MRJP3 has been reported to possess anti-inflammatory properties by the in vitro analysis, we investigated the anti-inflammatory effects of MRJP3 and its derived peptides both in vitro and in vivo. Expression of both tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) mRNAs in lipopolysaccharides (LPS)-stimulated THP-1 cells was reduced by the addition of MRJP3 or its C-terminal tandem penta-peptide repeats (TPRs) sequence. In the herpes simplex virus type 1 (HSV-1)-induced herpes stromal keratitis (HSK) model mice, the instillation of TPRs reduced the disease scores and the expression levels of TNF-α and IL-6 in HSV-1-infected eyes. In addition, synthetic penta-peptides derived from TPRs reduced the expression of TNF-α and IL-6 both in the THP-1 cell cultures and in the HSK model mice. Our results indicated that MRJP3 TPRs would be useful in controlling inflammation.

INTRODUCTION

Royal Jelly (RJ) is traditionally known as a functional food that possesses versatile pharmacological properties, including anti-inflammatory activity.^1,2) RJ contains many proteins that have a variety of functions.³⁾ A protein family, named major royal jelly proteins (MRJPs), represents more than 80% of RJ proteins. Among the nine members of the family, MRJP3 possesses immune modulating and anti-inflammatory functions both in vitro and in vivo.^4,5) MRJP3 has unique penta-peptide repeats at its C-terminus, which consist of highly basic residues, while the domains other than C-terminus are well conserved among the MRJP family of proteins.⁶⁾ The C-terminus of MRJP3 is made of 19 tandem penta-peptide repeats (TPRs). The TPRs form a complex with RNA, resulting in extracellular ribonucleoprotein, which protects RNA from degradation.⁷⁾ Recently, we have reported that TPRs also promote cell proliferation.⁸⁾

Inflammation is induced by microbial infection or tissue damage, and is characterized by the initiation and resolution phases.^9,10) Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β, are involved in the initiation of inflammatory responses. As these cytokines play important roles in the development and progression of several inflammatory diseases,¹¹⁾ the manipulation of these cytokine functions could provide therapeutic strategies for such inflammatory diseases.

In this report, we focused on the anti-inflammatory function of TPRs. The glutathione-S-transferase (GST)-fusion protein of TPRs and its trypsin-digested products reduced the expression of TNF-α and IL-6 in lipopolysaccharides (LPS)-stimulated THP-1 cells. In addition, the expression levels of pro-inflammatory cytokines were reduced by the addition of TPRs in the herpes simplex virus-infected mice model. The penta-peptide sequences of TPRs are classified into three patterns; i.e., QNAxN, QNxN[K/R], and RNGNR. Synthetic penta-peptides that have an NR or NK dipeptide sequence at their C-terminal end demonstrated the ability to reduce the expression of pro-inflammatory cytokines.

MATERIALS AND METHODS

Cells, Reagents, and Antibodies

Human monocyte-derived cell line THP-1 was cultured in RPMI containing 10% fetal bovine serum (FBS). The culture medium was supplemented with penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, U.S.A.). To stimulate THP-1 cells, lipopolysaccharide from Escherichia coli (E. coli) serotype O55:B5 (LPS, Sigma-Aldrich, St. Louis, MO, U.S.A.) was added to the culture medium at a final concentration of 1 µg/mL (1250 EU/µg).

Proteins and Peptides

GST-MRJP3 fusion proteins were purified from E. coli essentially as described previously.⁸⁾ In brief, E. coli strains harboring the plasmid for the expression of GST-fusion proteins (Fig. 1A) were cultured in the presence of Isopropyl-β-D-thiogalactopyranoside (IPTG) at 25 °C overnight, collected by centrifugation, resuspended in phosphate buffered saline (PBS), and sonicated using a BioRuptor sonicator (Sonic Bio, Kanagawa, Japan). The GST-fusion proteins were purified using Glutathione-Sepharose 4B beads (GE Healthcare, Chicago, IL, U.S.A.). The purified proteins were dialyzed against PBS at 4 °C overnight, and, if necessary, digested by trypsin as described previously.⁸⁾ Purified MRJP3 protein was kindly provided by Dr. Yonekura (Ibaraki Univ.).

Fig. 1. The Constructs of GST-Fusion Proteins and Amino-Acid Sequence of TPR Region

(A) The schematic representation of GST-TRP6 and GST-TPR19. TPRs consist of 5 copies of QNAxN (gray boxes), 13 copies of QNxN[K/R] (hatched boxes), and one copy of RNGNR (dotted box). The amino acid (aa) sequence 401–454 (for TPR6) or 401–519 (for TPR19) of MRJP3 is expressed as GST fusion proteins. Numbers indicate the aa positions of the MRJP3 protein. (B) The amino acid sequence of the TPR19 region. The dotted lines indicate the TPR6 sequence. The cleavage sites for trypsin treatment are indicated with arrowheads. Numbers indicate the aa positions of the MRJP3 protein.

Penta-peptides, QNADN, QNDNK, QNGNR, and RNGNR, which constitute the TPR sequence of MRJP3, were synthesized as a C-terminal-free carboxylic acid form with >95% HPLC purity by Biologica Co., Ltd. (Nagoya, Japan). The QNGNR peptide used for the animal experiment was synthesized by the standard Fmoc solid-phase synthetic method with C-terminal amides. After cleavage from the resin, the peptide was purified to >95% purity by HPLC (Agilent Technologies, Santa Clara, CA, U.S.A.) with an Inertsil SIL-100A column (GL Sciences Inc., Tokyo, Japan).

Quantitative RT-PCR (qRT-PCR)

To analyze the expression of pro-inflammatory cytokines in vitro, THP-1 cells were stimulated with 1 µg/mL of LPS, followed by the addition of proteins (250 µg/mL) or synthetic peptides (100 µM) and incubation at 37 °C. At 4 h post-treatment, total RNA samples were extracted with TRIZol reagent (Thermo Fisher Scientific). For the analysis of expression kinetics, samples were collected at 4, 8, and 24 h post-treatment. The extracted RNA samples were reverse transcribed using a PrimeScript RT reagent kit (TaKaRa Bio, Shiga, Japan), and the expression levels of human TNF-α, IL-6, and β-actin were determined by quantitative PCR with the primers shown in the Supplementary Table.

In the case of herpes stromal keratitis (HSK) model mice, total RNA samples were prepared from the eyeballs that were inoculated with herpes simplex virus type 1 (HSV-1), and the expression levels of TNF-α, IL-6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were analyzed as described above except for the primers for mouse targets (Supplementary Table).

Luciferase Reporter Assay

HEK293T cells were transfected with the expression plasmids for LPS receptors (p3 × FLAG-CMV9-mTLR4, p3 × FLAG-CMV9-mCD14, and pBudcE4.1-mMD2)¹²⁾ and reporter plasmids (pGL4.32 and pRL-TK, Promega, Madison, WI, U.S.A.). To determine the AP-1 signaling, cells were transfected with LPS receptor plasmids, pAP1(PMA)-TA-Luc, and pRL-TK. At 24 h post-transfection, cells were treated with LPS (1 µg/mL) and/or peptide B2 (100 µM). After 24 h post-treatment, the firefly and Renilla luciferase activities were analyzed with a Dual-Glo luciferase assay kit (Promega) as described previously.¹³⁾

Animal Study

The experimental protocol of this study was approved prior to the study’s commencement by the Animal Care and Use Committee of Gifu Pharmaceutical University and Gifu University. All animal experiments were performed accordance with the experimental animal guidelines of institutions. Female ICR mice at 5 weeks of age (Japan SLC, Inc., Shizuoka, Japan) were ocularly infected with HSV-1 strain F under anesthesia to develop the HSK model. Virus solutions were prepared as 1 × 10⁶ plaque forming unit (pfu)/head/5 µL with PBS, PBS-GST (1 mg/mL), or PBS-GST-TPR19 (1 mg/mL). Five micro-liters of each virus solution was dropped into the right eye which had been scarified with a 27-gauge needle (day 0). Additionally, PBS, PBS-GST, PBS-GST-TPR19, or the synthetic peptide QNGNR (1 mg/mL, 5 µL/head) was instilled into the infected eyes under inhalant anesthesia (Isoflurane, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for the next 4 d (from day 1 to 4 post-infection). In experiment 1 (7–9 mice per group), the severities of HSK and periocular skin disease were scored blindly by three independent evaluators based on the scoring systems described previously.^14,15) The HSK scores were as follows: 0, normal cornea; 1, mild corneal haze; 2, moderate corneal opacity or scarring; 3, severe corneal opacity, iris not visible; 4, opaque cornea; and 5, necrotizing stromal keratitis. The periocular skin disease scores were as follows: 0, no lesions; 1, minimal eyelid swelling; 2, moderate eyelid swelling accompanied by crusty ocular discharge; 3, severe eyelid swelling and moderate hair loss in periocular skin; and 4, severe swelling with eyes crusted shut, severe periocular hair loos and skin lesions. In experiment 2, for RNA preparation, the infected eyeballs were dissected at 3 d post-infection (dpi) from 5 mice per group. In experiment 3, for evaluation of viral growth, the infected eyeballs, trigeminal ganglia (TG), and brains of 6 mice per group were collected at 1 and 5 dpi into Dulbecco’s modified Eagle’s medium (DMEM) containing 1% FBS and frozen at −80 °C until use as described previously.¹⁵⁾ After freeze-thawing, the samples were sonicated with Biorupter (Sonic Bio), centrifuged to clarify the debris, and serially diluted for plaque assay in Vero cells.

Statistical Analysis

Data were analyzed by unpaired Student’s t-test or one-way ANOVA followed by Dunnett’s or Tukey’s test using GraphPad Prism 8 (GraphPad Software, San Diego, CA, U.S.A.). p-Values less than 0.05 were considered significant.

RESULTS

MRJP3 TPRs Reduced the Expression of Inflammatory Cytokines

To examine the anti-inflammatory properties of TPRs, GST or GST-TPRs (TPR6 and TPR19, Figs. 1A, B) were added to LPS-treated THP-1 cells. At 4 h post-treatment, mRNAs were purified from the cells and analyzed with qRT-PCR. Treatment with GST-TPR19 and MRJP3, but not with GST or GST-TPR6, reduced the expression levels of IL-6 and TNF-α (Fig. 2A). The trypsin-digested GST-TPR6, GST-TPR19, and MRJP3, but not the digested GST, were reduced the expression levels of IL-6 and TNF-α (Fig. 2B). These results indicated that the TPRs and peptides derived from them possessed anti-inflammatory activities.

Fig. 2. The Reduction of TNF-α and IL-6 Expression by MRJP3 or TPRs in Cell Cultures

THP-1 cells were cultured in the presence of 1 µg/mL of LPS along with 100 µg/mL of GST-fusion proteins or MRJP3 that were undigested (A) or digested with trypsin (B). At 4 h post-treatment, the expression levels of IL-6 and TNF-α were measured by qRT-PCR. Means ± standard error of the means (S.E.M.s). of the gene expression levels in the cells treated with the indicated protein relative to those of the cells treated with PBS in three independent experiments are shown. The p-values were determined by one-way ANOVA followed by Dunnett’s multiple comparison test against the GST group. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Anti-inflammatory Properties of TPR19 in a Herpes Simplex Virus Keratitis Model

To demonstrate the effects of TPR19 on virally induced inflammation, we employed the murine model of HSK in which, after ocular inoculation of HSV-1, viral replication in the eyes induces inflammatory responses such as keratitis and blepharitis. Using this model, the anti-inflammatory activities of GST-TPR19 were evaluated by instillation of the indicated materials into the HSV-1-infected eyes once a day for 5 d. The instillation of GST-TPR19 reduced the HSK and periocular disease scores at 6 dpi (Fig. 3A). To determine the expression levels of pro-inflammatory cytokines, infected eyes were collected at 3 dpi and analyzed with qRT-PCR. As shown in Fig. 3B, the expression levels of TNF-α and IL-6 mRNA were substantially reduced by GST-TPR19 in comparison with those of the PBS or GST group. However, the instillation of GST-TPR19 did not impair viral replication in the eyes, TG, or brain (Fig. 3C). These results indicated that TPRs induced anti-inflammatory activity in the absence of antiviral effects in vivo.

Fig. 3. Effect of Eye Instillation of TPRs in Ocularly Infected Mice

(A) HSK disease scores in TPRs-treated mice. The right eye of each 5-week-old female ICR mouse was infected ocularly with 1 × 10⁶ pfu of HSV-1(F). The infected mice were then instilled with PBS, GST, or GST-TPR19 (1 mg/mL, 5 µL) once a day for 5 d. HSK and periocular disease scores were obtained at 6 dpi. Each data point represents the HSK score from an individual mouse. Horizontal bars represent the mean ± S.E.M. for each group (n = 8 or 9). Significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, and *** p < 0.001. (B) The amounts of TNF-α and IL-6 mRNA in HSV-1-infected eyes. The amounts of mRNA purified from the eyes of HSV-1-infected mice at 3 dpi were analyzed by qRT-PCR. Horizontal bars represent the mean ± S.E.M. for each group (n = 5). Significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, and *** p < 0.001. (C) The infected eyes, trigeminal ganglia, and brains were collected at 1 or 5 dpi. The viral yields in these tissues were assayed on Vero cells (mean ± S.E.M., n = 6).

Anti-inflammatory Properties of TPRs-Derived Peptides

The reduction in the expression levels of pro-inflammatory cytokines by the trypsin-digested TPR19 suggests that the penta-peptide is the determinant of the anti-inflammatory properties of MRJP3. To examine this hypothesis, we used four synthetic penta-peptides derived from the TPR sequence shown in Fig. 4. These peptides were added to LPS-treated THP-1 cells at a final concentration of 100 µM. As shown in Fig. 4B, peptides B1, B2, and C reduced the expression levels of TNF-α and IL-6. Therefore, the N and K/R residues at positions 4 and 5, respectively, of the penta-peptides are important for the anti-inflammatory function of TPRs. The expression TNF-α mRNA, which induced at 4 h after the addition of LPS, was depressed by the peptide B2 treatment (Fig. 4C).

Fig. 4. The Reduction of TNF-α and IL-6 Expression by Synthetic Peptides in Vitro

(A) The sequences of the penta-peptides used in this experiment. (B) THP-1 cells were treated with 100 µM of synthetic peptides in the presence of 1 µg/mL of LPS. At 4 h post-treatment, the expression levels of IL-6 and TNF-α were measured by qRT-PCR. Mean ± S.E.M.s of the gene expression levels in the cells treated with the indicated peptides relative to those with PBS in three independent experiments are shown. The p-values were determined by one-way ANOVA followed by Dunnett’s multiple comparison test against the PBS group. ** p < 0.01 and *** p < 0.001. (C) The kinetics of TNF-α mRNA expression. THP-1 cells were collected at 4, 8, or 24 h after treatment with LPS (1 µg/mL) and synthetic peptide B2 (100 µM) (open circles) or with LPS alone (closed circles). The expression of TNF-α mRNA was analyzed by qRT-PCR (means ± S.E.M.s., n = 5 or 6). The p-values were determined by one-way ANOVA followed by Tukey’s multiple comparison test. **** p < 0.0001. (D) Time-of-addition assay of peptide B2. THP-1 cells were treated with LPS and peptide B2 as indicated in schema. At 4 h post-treatment with LPS, the cells were harvested, and the mRNA expression levels of TNF-α were analyzed by qRT-PCR (means ± S.E.M.s., n = 3). The p-values were determined by one-way ANOVA followed by Dunnett’s multiple comparison test against the PBS group. ** p < 0.01 and *** p < 0.001. (E) Dose dependency of synthetic peptides. THP-1 cells were treated with LPS (1 µg/mL) and synthetic peptides at the indicated concentrations. At 4 h post-treatment, the cells were harvested, and the mRNA expression levels of TNF-α were analyzed by qRT-PCR (means ± S.E.M.s., n = 5 or 6). The p-values were determined by Kruskal–Wallis test against the PBS group. * p < 0.05 and ** p < 0.01.

To elucidate the inhibitory mechanisms of TPR-derived peptides, we performed a time-of-addition experiment using synthetic peptide B2 as shown in Fig. 4D. The expression of TNF-α was prevented by the pre-treatment with peptide B2 30 min ahead of the LPS treatment. The peptide B2 treatment initiated at 30 min, but not at 60 min, after the addition of LPS was still effective.

To determine the functional doses of TPR-derived peptides, THP-1 cells were treated with several concentrations of synthetic peptides (Fig. 4E). Peptides B1 and B2 at 100 µM but not at 10 µM significantly inhibited the expression of TNF-α. Peptide C also showed inhibitory effect at 100 µM, although it was not statistically significant.

Nuclear factor-kappa B (NF-κB) signaling is the major signaling pathway which is activated by the addition of LPS to express the pro-inflammatory cytokines.¹⁶⁾ As HEK293T cells lack the expression of LPS receptors (TLR4, MD2, and CD14), the receptor plasmids were transfected along with the reporter plasmids to determine the inhibitory signaling of peptide B2 (100 µM) as described in Materials and Methods. As shown in Fig. 5A, the NF-κB signaling was impaired by the peptide B2 treatment, indicating that TPR peptides inhibited the NF-κB signaling pathway. In contrast, the AP-1 signaling was not altered by the peptide B2 treatment (Fig. 5B).

Fig. 5. The Inhibition of NF-κB Signaling by TPR Peptides

HEK293T cells transfected with LPS receptors (TLR4, CD14, and MD2) and reporter plasmids were treated with LPS (1 µg/mL) and peptide B2 (100 µM) for 24 h and analyzed as described in Materials and Methods. The means and S.E.Ms. (n = 3) of ratios of Firefly/Renilla luciferase activities (ratio of activities) of NF-κB (A) and AP-1 (B) signaling are shown. The p-values were determined by one-way ANOVA followed by Tukey’s multiple comparison test against the PBS group. **** p < 0.0001.

To evaluate the anti-inflammatory effects of the penta-peptides in vivo, peptide B2 was instilled into the eyes of HSV-1-infected mice. At 3 dpi, the expression levels of TNF-α and IL-6 mRNA were significantly reduced in the peptide B2-instilled group (Fig. 6A). The synthetic peptide B2 demonstrated anti-inflammatory activity in vivo, although the reduction in disease scores was not significant (Fig. 6B).

Fig. 6. The Reduction of TNF-α and IL-6 Expression by Synthetic Peptides in HSK Model

(A) The expression of TNF-α and IL-6 mRNA in peptide B2-treated eyes. Five-week-old female ICR mice were infected ocularly with 1 × 10⁶ pfu/eye of HSV-1(F). PBS or peptide B2 (1 mg/mL, 5 µL/d) was then instilled once a day for 3 d. The expression of mRNA in the eyes of HSV-1-infected mice at 3 dpi was analyzed by qRT-PCR. Horizontal bars represent the mean ± S.E.M. for each group (n = 5). Significance was determined by Student’s t-test. * p < 0.05. (B) HSK disease scores in the peptide B2-treated mice. HSK model mice were instilled with PBS or peptide B2 (1 mg/mL, 5 µL/d) once a day for 5 d. The HSK and periocular disease scores were obtained at 6 dpi. Each data point represents the HSK or periocular score from an individual mouse. Horizontal bars represent the mean ± S.E.M. for each group (n = 7 or 8). Significance was determined by Student’s t-test.

DISCUSSION

In this study, we found that TPRs and the synthetic peptides prepared from one of the major protein constituents of RJ decreased the expression of pro-inflammatory cytokines both in vitro and in vivo. TPR-derived peptides prevented the activation of NF-κB signaling stimulated by the LPS treatment. Furthermore, the instillation of TPRs reduced the disease severity in HSK model mice. Particular amino acid (aa) sequences; namely, N and K/R residues at aa positions 4 and 5, were important for such anti-inflammatory properties, suggesting that the penta-peptide is the minimum unit for the anti-inflammatory function of MRJP3.

The instillation of TPRs reduced the expression levels of TNF-α and IL-6, but did not inhibit viral replication in HSV-1 infected mice. It is known that the development of HSK correlates with the expression of IL-6 but not with the HSV-1 titers in the cornea in the mouse model.¹⁷⁾ HSK is characterized by chronic inflammation of the corneal stroma due to cell infiltration and neovascularization.¹⁸⁾ Infection of the cornea with HSV-1 induces innate immune responses, including the expression of pro-inflammatory cytokines, which leads to cell infiltration and neovascularization.^19,20) Although it is necessary to elucidate the precise mechanism(s), we hypothesize that TPR reduced the HSK disease severity by suppression of pro-inflammatory cytokine expression.

MRJP3 has the ability to modulate immune responses.^4,5) The expression levels of pro-inflammatory cytokines were reduced by treatment with MRJP3, GST-TPR19 and the trypsin-digested products, indicating that the TPRs are important for this modulation of immune responses. As the synthetic peptides QNDNK, QNGNR, and RNGNR reduced the expression levels of pro-inflammatory cytokines, these sequences are thought to be responsible for anti-inflammatory properties of TPR19. It is noteworthy that the trypsin digestion of GST-TPR6 bestowed anti-inflammatory properties (Fig. 2), which may indicate the presence of an additional anti-inflammatory peptide(s) in the aa 401–425 region of MRJP3.

Recently, peptide drugs have been envisaged as candidates for novel breakthrough medicines.²¹⁾ TPR-derived peptides have the potential to act as anti-inflammatory agents. The control of inflammation is important for the maintenance of a healthy lifestyle, as inflammation is involved in the development and exacerbation of many diseases, including cancer, neurodegenerative diseases, and infections.^22–24) Although the mRNA expression of pro-inflammatory cytokines was reduced, the disease scores were not significantly improved in the eyes instilled with peptide QNGNR (Figs. 6A, B). In addition, the inhibitory effects in cells were observed only at the high concentration of peptides (Fig. 4E). These results suggested that the need of further optimization of the peptide sequence itself, modification of the terminus forms of the peptides, and/or an increase in peptide stability with the aid of drug delivery methods.

In conclusion, this study demonstrated that particular penta-peptides derived from MRJP3 are strong candidates as agents for the prevention of inflammation and the promotion of a healthy lifestyle.

Acknowledgments

The plasmids for LPS receptors were kindly provided from Drs. Y. Adachi and N. Ohno (Tokyo Pharmaceutical University, Japan).

This work was partly supported by funding from API Co., Ltd. (N.I.) and a Grant from Gifu Pharmaceutical University (T.K.).

Conflict of Interest

NI received a collaborative research fund from Api Co., Ltd., a company that handles bee derived products and functional foods. However, the company was not involved in the collection, management, analysis, or interpretation of the data, or in any processes related to manuscript preparation. All other authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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